Metalloporphyrins as chemical mimics of cytochrome P-450 systems
The liver, in humans and animals, naturally metabolizes most drug molecules through
oxidative mechanisms into metabolites. The formation of these metabolites is catalyzed
predominantly by cytochrome containing enzymes bound with a heme cofactor. The most familiar of
these enzymes responsible for catalyzing a range of biological oxidations is the P-450 monooxygenase. In all of these heme-containing proteins which includes catalases, peroxidases and
ligninases, the proteins are activated by hydrogen peroxide, and catalysis is initiated via a two-electron
oxidation of the ferric resting state to an oxoferryl porphyrin cation radical (I).
H2O2
Fe(III)porphyrin
H2O
O
+ .
Fe(IV)porphyrin
I
While the oxidation state for the cytochromes P-450 has yet to be characterized, most of their
reactions and those of the biomimetic analogs can be accounted for by an oxygen transfer from (I) to
a variety of substrates to give characteristic reactions such as hydroxylation, epoxidation and heteroatom
oxidation. Other products resulting from hydroxyl and hydroperoxyl radicals have also been detected.
The metabolic processes in vivo contribute substantially towards the efficacy, side effects and toxicity
of the pharmaceutical entity. These factors are key in the success or failure of a clinical candidate
therefore the metabolic processes of drug molecules in the body are always the subject of
intense scrutiny in pharmaceutical companies.
Toxicological and pharmacological studies on the metabolites form a crucial segment in the
identification of a clinical candidate. Traditionally pharmacologists have been involved in the isolation
and identification of the metabolites of a drug. It is imperative that metabolite studies are conducted
early in the drug development process.
Current issues:
Several issues of contention have come to attention with the use of biological systems in
studying drug metabolism:
(i)
In vitro studies produce very small quantities of the primary metabolites, which are often
hydrophilic and difficult to isolate.
(ii)
Animal studies necessitate the sacrifice of animals and are extremely expensive to conduct.
Liver slice preparations are of variable potency; it is difficult to quantitate the precise
stoichiometry of the oxidant.
(iii)
Pharmacologists have no prior knowledge of the metabolite structure they need to seek.
(iv)
Many of the metabolites are not amenable to organic synthesis by conventional routes.
We therefore turned to the metalloporphyrins as mimics of the in vivo metabolic processes.
The oxometalloporphyrins:
Synthetic metalloporphyrins have recently received a lot of attention as mimics of numerous
enzymes, specifically 10 models for peroxidases and particularly the ligninases.
Metalloporphyrins have also found utility as model systems for studies of the oxidative
metabolism of drugs. A detailed study of the metabolism of lidocaine has been reported as have
preliminary studies on the use of metalloporphyrins as chemical mimics of cytochrome P-450
systems.78
The first synthetic metalloporphyrins were found to be oxidatively labile. Few catalytic
turnovers were seen due to the rapid destruction of the porphyrin macrocycle. Dolphin showed
that the introduction of halogen groups onto the aryl moeity (of meso-tetraarylporphyrins) and
on the -pyrrolic positions of the porphyrins increased the turnover of catalytic reactions by
decreasing the rate of porphyrin destruction. In addition, the combined electronegativities of the
halogen substituents are transmitted to the metal atom making the corresponding oxocomplexes more electron deficient and thus a more effective oxidation catalyst.
Application of the Methodology to Selected Drugs:
We have recently applied this technique to study the results of oxidative metabolism on several
drugs. Reactions using our metalloporphyrins were conducted in methylene chloride/water or
water/acetone to nitrile (80:20, v/v) at ambient temperatures. Iodosobenzene, cumene
hydroperoxide, hydrogen peroxide or sodium hypochlorite were used as the source of
exogenous oxygen. Products were separated by HPLC and identified by comparing authentic
reference samples of the major metabolites of these drugs previously isolated from the urine of
rats or characterized from rat liver microsomal incubations.
Scheme P
Cl
Cl
Cl
FeTPPF20
HO
O
N Me
N H
HO
HO
HO
+
cumene hydroperoxide
O
Cl
N Me
N Me
O
O
O
CH2Cl2
OH
cis and trans
Oxidation of Odapipam (Anti-psychosis Drug)
Scheme Q
O
O
O
OH
O
O
O
O
O
O
O
FeTPPCl8Br8
+
+
+
PhIO
HO
N
N
N
N
OH
O
Oxidation of ABT-200 (an Anti-depressant Drug)
Scheme R
Me
N
Me
Me
O
FeTPPCl8Br8
N
N
O N
Me
PhIO, CH2Cl2
O N
H
O N
Me
Me
O
Me
N
O N
Me
O N
H
O N
Oxidation of Cholinergic Channel Activator for the treatment of Alzheimer's Disease
Scheme S
OH
N
O
N
Me N
Me
Me
FeTPPCl8Cl8(SO3H)4
PhIO
H2O/ CH3CN (80:20)
N
O
Me N
H
Me
N Me
+
N
O
Me N
Me
Me
2%
Me 3%
N
O
H2N
N Me
Me N
Me
4%
N
O
+
N Me
+
O
N Me
Me
43-53%
N
HN
+
CH2OH
O
N
N
Me N
O Me
Me
CH2OH
1%
N Me
Me
CHO
12-17%
Selective Oxidation of Aminopyrine
The oxidation of aminopyrene was particularly interesting in that new, hitherto unobserved
metabolites were detected. The variation of the reaction conditions led to the synthesis of selected
metabolites in increased yields.
Scheme T
OH
N
O
Me N
Me
N
.HCl
Fe(III)Cl8Cl8TPP(SO3H)4
O
PhIO
MeOH/ CH3CN (1:20)
Me N
Me
N
N Me
N Me
Me N
Me
Me
16%
Me
N
O
Fe(III)Cl8Cl8TPP(SO3H)4
N
O
N Me
PhIO
pH = 1 H2O/ CH3CN
Me
Me N
CH2OH
Me
78%
Me
Fe(III)Cl8Cl8TPP(SO3H)4
O
H2N
N
N Me
N
O
PhIO
pH = 7 phosphate/ CH3CN
Me
HO NH
N Me
Me
30%
Selective Oxidation of Aminopyrine
This was also exemplified by the oxidation of lidocaine; two new oxidation products were observed.
Scheme U
CH3 H
CH3 H
N
N
O
CH3
CH3 H
Fe(III)Cl8Cl8TPP(SO3H)4
N
PhIO
pH = 7 phosphate/ CH3CN
O
CH3
N
CH3
CH3
N
CH3
O
CH3
34%
HO
O
N
N
CH3
O
CH3
CH3
O
CH3
NH
CH3
CH3
Fe(III)Cl8Cl8TPP(SO3H)4
PhIO
MeOH/CH3CN (1:20)
N
O
CH3
13%
CH3
CH3
15%
CH3 H
HO
N
+
4%
Cl
H
CH3 H
O
CH3
N
H
6%
CH2 H
N
CH3 H
N
+
H
CH3 H
N
CH3
HO
N
N
+
CH3
Oxidation of Lidocaine
These two oxidation products have escaped detection in all previous studies.
O
CH3
2%
CH3
CH3
It is useful to note that these are rare examples of porphyrin-mediated oxidation of sophisticated
pharmaceutical entities. The reactions described above are generally applicable and have been used in our
laboratories to achieve hydroxylation and N-demethylation reactions on numerous other substrates. The
hydroxyl metabolite and the desmethyl derivative were usually difficult to synthesize by conventional
organic chemistry methods.
This approach affords an efficient method for the systematic preparation and identification of the entire
spectrum of metabolites from a chosen drug. We can envision the screening of a series of compounds by
reacting them with different permutations of a metalloporphyrin, co-oxidant and a suitable solvent. Reaction
conditions can be refined to produce the maximum number of metabolites. This logically leads to
subsequent scaled-up optimal processes. The oxidation products can be separated and subjected to
toxicological, pathological, histopathalogical, or gentoxic testing.